HEK293 cells transfected with human MATE1, MATE2-K, or vector control were established by
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1 SUPPLEMENTAL DIGITAL CONTENT METHODS In Vitro Metformin Transport Studies Effect of Dolutegravir on Metformin Transport by MATE1 and MATE2-K HEK293 cells transfected with human MATE1, MATE2-K, or vector control were established by Sekisui Medical Co., Ltd. (Naka-gun, Ibaraki, Japan). Cells were cultured in 75-cm 2 bottom flasks and subjected to passage every 3 to 4 days. The culture medium was exchanged every 4 days. MATE1 cells were seeded in collagen I-coated 24-well plates at a density of cells/well and incubated (37 C, 5% CO 2) for 2 days. For MATE2-K, HEK293 cells were seeded in collagen I-coated 12-well plates at a density of cells/well and incubated (37 C, 5% CO 2) for 1 day; cells were then transfected with MATE2-K cdna and incubated for an additional 2 days. Uptake experiments were initiated by pre-incubation (15 minutes, 37 C) with Gibco Hank s Balanced Salt Solution (HBSS; ph 8.5 to invert MATE transport directionality inward) containing dolutegravir or cimetidine (0-100 µm). After pre-incubation, cells were incubated (5 minutes, 37 C) with HBSS (ph 8.5) solutions containing 10 µm [ 14 C]metformin, dolutegravir, or cimetidine (0-100 µm). Cellular uptake of [ 14 C]metformin was quantified by radiometric detection; protein content was determined by Pierce BCA Protein Assay Kit per the manufacturer s instructions. Effect of Dolutegravir on Metformin Transport by PMAT and OCT3 MDCK-II cells transfected with human PMAT, OCT3, or vector control were established by Optivia Biotechnology, Inc (Menlo Park, CA). MDCK-II cells were maintained in Dulbecco s Modified Eagle Medium (DMEM) with low glucose, low sodium bicarbonate, and 10% fetal bovine serum (FBS). Cells between passage 7 and 30 were seeded at a density of 6± cells/well on 96-well transwell membrane plates 1 day before transfection; transport assays were carried out following 2-day incubation (37 C, 5% CO 2) after transfection.
2 Uptake experiments were initiated by pre-incubation in both apical and basolateral chambers (15 minutes, 37 C, orbital shaking at 60 rpm) with HBSS containing inhibitors dolutegravir (0-100 µm), quinidine (1 mm), decynium-22 (100 µm), or verapamil (0-100 µm). After pre-incubation, cells were incubated in the basolateral chambers (5 minutes, 37 C, 60 rpm) with HBSS solutions containing inhibitors and probe substrates: 10 µm [ 14 C]metformin for both PMAT and OCT3; 10 µm [ 14 C]1-methyl- 4-phenylpyridinium (MPP+) for PMAT, 2 µm [ 14 C]MPP+ for OCT3, and 100 µm [ 14 C]tetraethylammonium for OCT3. Cellular uptake of probe substrate was determined by radiometric detection. Effect of Dolutegravir on Paracellular Permeability and Cellular Uptake of Metformin Caco-2 cells (wild type C2BBe1, ATCC catalog # CRL-2102) were received from Sigma- Aldrich, Inc (St. Louis, MO) as a high throughput assay ready plate on day 16 or 17 of culture. Plates were Millicell 24-cell culture multi-well inserts with polycarbonate membranes, 0.4 µm pore size, and 0.7 cm 2 surface area. Pre-seeded plates were unpacked and processed according to the vendor s instructions. Briefly, plates were left at room temperature for 24 to 48 hours then elevated to 37 C, 5% CO 2 in a cell culture incubator for 4 hours to liquefy the shipping medium and to allow a culture medium change (DMEM, 20% FBS, supplemented with 2 mm L-glutamine). Following the initial media change, plates were incubated at 37 C with 5% CO 2 and culture medium was replaced every 2 to 3 days. Plates were used prior to day 25 of culture as recommended by the vendor. Effect of dolutegravir on Lucifer yellow paracellular flux: basolateral and apical chamber incubation solutions were transport medium (DMEM supplemented with L-glutamine, 25 mm HEPES, pyridoxine HCl, but without sodium pyruvate and phenol red, ph 7.4) containing dolutegravir ( µm) for the first 15 minutes. For the subsequent 90 minutes, the apical incubation solutions were transport medium containing dolutegravir ( µm) and 100 µm Lucifer yellow. In a separate set of experiments, basolateral-to-apical flux of Lucifer yellow was determined in confluent MDCKII cell monolayers in the absence or presence of dolutegravir ( µm).
3 Effect of dolutegravir absorptive (apical-to-basolateral) flux of [ 14 C]metformin: basolateral and apical chamber incubation solutions were transport medium containing dolutegravir ( µm) for the first 15 minutes. For the subsequent 90 minutes, the apical incubation solutions were transport medium containing dolutegravir ( µm) and [ 14 C]metformin ( mm). Effect of dolutegravir on the cellular uptake of [ 14 C]metformin: basolateral and apical chamber incubation solutions were transport medium containing dolutegravir ( µm) for the first 15 minutes. Subsequently, the apical incubation solutions were transport medium containing dolutegravir ( µm) and [ 14 C]metformin ( mm). Cell monolayers were incubated at 37 C with shaking in the time-linear range for metformin uptake (10 minutes at 0.05 mm, 2.5 minutes at 0.5 and 5 mm). Lucifer yellow concentrations were measured by fluorescence spectrophotometry. [ 14 C]metformin was quantified by radiometric detection; protein content was determined by Thermo Scientific Pierce BCA Protein Assay Kit per the manufacturer s instructions.
4 SUPPLEMENTAL DIGITAL CONTENT RESULTS In Vitro Metformin Transport Studies Effect of Dolutegravir on Metformin Transport by MATE1 and MATE2-K Dolutegravir (GSK ) was not a clinically relevant inhibitor of [ 14 C]metformin transport by MATE1 (IC 50 = 6.3±1.1 μm) and MATE2-K (IC 50 = 24.8±4.7 μm) (Supplemental Figure 1). The positive control MATE inhibitor, cimetidine, inhibited MATE1- and MATE2-K-mediated [ 14 C]metformin transport with IC 50 values of 2.8±0.5 and 3.6±0.6 μm, respectively (Supplemental Figure 2). Cell viability in control, MATE1, and MATE2-K-expressing HEK293 cells in the presence of dolutegravir ranged from 99% to 111% of vehicle control for up to 100 μm dolutegravir (data not shown). Effect of Dolutegravir on Metformin Transport by PMAT and OCT3 Dolutegravir ( 100 μm) did not inhibit [ 14 C]MPP+ uptake by PMAT or OCT3 (Supplemental Figure 3). Likewise, dolutegravir ( 100 μm) did not inhibit [ 14 C]metformin uptake by PMAT, and it did not inhibit OCT3 at concentrations 10 μm, while 10% to 25% inhibition of [ 14 C]metformin transport by OCT3 was noted at 30 to 100 μm dolutegravir concentrations (Supplemental Figure 4). Positive control inhibitors, 100 μm decynium-22 for PMAT and 1 mm quinidine for OCT3, inhibited PMAT and OCT3 transport activities by 78% to 88% and 92% to 95%, respectively. In addition, the positive control OCT3 inhibitor, verapamil, inhibited transport of 100 μm [ 14 C]tetraethylammonium via OCT3, with an IC 50 of 2.94 µm, and 95.8% inhibition at 100 µm verapamil (data not shown). Effect of Dolutegravir on Paracellular Permeability and Cellular Uptake of Metformin Dolutegravir ( µm) did not increase the paracellular permeability of Lucifer yellow in a concentration-dependent manner in Caco-2 cell monolayers, with mean absorptive (apical-to-basolateral) permeability of 7.5 nm/sec and 14.2 nm/sec at 0.3 µm and 1 to 100 µm dolutegravir, respectively. Likewise, in MDCKII cell monolayers Lucifer yellow basolateral-to-apical Lucifer yellow permeability was comparable in the absence and presence of ( µm) dolutegravir (11.1±5.0 vs 9.4±4.2 nm/sec, respectively). Finally, dolutegravir ( 100 µm) did not increase the absorptive (apical-to-basolateral) flux
5 of metformin in Caco-2 cell monolayers (Supplemental Table 1). Dolutegravir ( µm) did not affect the cellular uptake of metformin (0.05, 0.5, 5 mm) into Caco-2 monolayers (Supplemental Table 2).
6 Supplemental Table 1. Effect of Dolutegravir (GSK ) on [ 14 C]Metformin Flux in Caco-2 Cell Monolayers Metformin Concentration GSK Concentration (μm) Rate A B (pmoles/min/cm 2 ) A B Mass Balance (%) 0.05 mm [ 14 C]metformin 0 1.3± ± ±27 105± ± ± ±5.8 99± ±30 103± ±15 101± ±9.6 97± mm [ 14 C]metformin 0 17± ± ± ± ± ± ± ± ± ± ± ± ± ± mm [ 14 C]metformin 0 136± ± ± ± ± ± ± ± ± ± ± ± ± ±0.025
7 Supplemental Table 2. Effect of Dolutegravir (GSK ) on [ 14 C]Metformin Cellular Uptake in Caco-2 Cell Monolayers mm [ 14 C]Metformin 0.5 mm [ 14 C]Metformin 5 mm [ 14 C]Metformin (pmol/μg (pmol/μg (pmol/μg GSK (μm) protein) SD protein) SD protein) SD SD, standard deviation. Data are average of samples from triplicate wells.
8 Inhibitory effect of GSK on MATE1-mediated transport of metformin IC 50 value for the inhibition by GSK on MATE1-mediated transport of metformin MATE1-expressing cells Cleared volume (µl/mg protein) Concentration of GSK (µm) Slope factor: Concentration of GSK (µm) Each bar represents the mean ± SD of three samples. Substrate (concentration) Test compound Concentration (µm) Cleared volume (µl/mg protein) MATE1-expressing cells % of control IC 50 [ 14 C]Metformin GSK ± ± ± 1.11 (10 µm) ± ± Each value represents the mean ± SD of three samples. The IC 50 value represents the mean ± SE ± ± ± ± ± ± ± ± ± ± ± ± ± ±
9 Inhibitory effect of GSK on MATE2-K-mediated transport of metformin IC 50 value for the inhibition by GSK on MATE2-K-mediated transport of metformin 15 MATE2-K-expressing cells Cleared volume (µl/mg protein) Concentration of GSK (µm) Slope factor: Concentration of GSK (µm) Each bar represents the mean ± SD of three samples. Substrate (concentration) Test compound Concentration (µm) Cleared volume (µl/mg protein) MATE2-K-expressing cells % of control IC 50 [ 14 C]Metformin GSK ± ± ± 4.7 (10 µm) ± ± Each value represents the mean ± SD of three samples. The IC 50 value represents the mean ± SE ± ± ± ± ± ± ± ± ± ± ± ± ± ±
10 Inhibitory effect of cimetidine on MATE1-mediated transport of metformin IC 50 value for the inhibition by cimetidine on MATE1-mediated transport of metformin MATE1-expressing cells Cleared volume (µl/mg protein) Concentration of cimetidine (µm) Slope factor: 1.08 Concentration of cimetidine (µm) Each bar represents the mean ± SD of three samples. Substrate (concentration) Test compound Concentration (µm) Cleared volume (µl/mg protein) MATE1-expressing cells % of control IC 50 [ 14 C]Metformin Cimetidine ± ± ± 0.46 (10 µm) ± ± Each value represents the mean ± SD of three samples. The IC 50 value represents the mean ± SE ± ± ± ± ± ± ± ± ± ± ± ± ± ±
11 Inhibitory effect of cimetidine on MATE2-K-mediated transport of metformin IC 50 value for the inhibition by cimetidine on MATE2-K-mediated transport of metformin 15 MATE2-K-expressing cells Cleared volume (µl/mg protein) Concentration of cimetidine (µm) Slope factor: Concentration of cimetidine (µm) Each bar represents the mean ± SD of three samples. Substrate (concentration) Test compound Concentration (µm) Cleared volume (µl/mg protein) MATE2-K-expressing cells % of control IC 50 [ 14 C]Metformin Cimetidine ± ± ± 0.59 (10 µm) ± ± Each value represents the mean ± SD of three samples. The IC 50 value represents the mean ± SE ± ± ± ± ± ± ± ± ± ± ± ± ± ±
12 Inhibition of 2 M MPP+ uptake mediated by OCT3 Percent of Control (%) GSK (µm) 10 µm MPP+ uptake by PMAT: GSK (µm) Cellular (transporter) Cellular (control) Net Transporter Mediated Cellular Inhibition (%) ± µm Decynium µm MPP+ uptake by OCT3: GSK (µm) Cellular (transporter) Cellular (control) Net Transporter Mediated Cellular Inhibition (%) ± um um um um um um um um um um mM Quinidine
13 Inhibition of 10 µm Metformin uptake mediated by OCT3 120 Percent of Control (%) GSK (µm) 10 µm Metformin uptake by PMAT: GSK (µm) Cellular (transporter) Cellular (control) Net Transporter Mediated Cellular Inhibition (%) ± µm Decynium µm Metformin uptake by OCT3: GSK (µm) Cellular (transporter) Cellular (control) Net Transporter Mediated Cellular Inhibition (%) ± mM Quinidine
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